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  • 专利说明
  • 权利要求
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    • 专利摘要:
    Synthesis of fluorescent derivatives with applications in the labeling of proteins, cells and nucleic acids. The present invention details the synthesis of fluorescent compounds capable of binding to biological molecules: proteins, nucleic acids or cells and their applications in protein labeling, nucleic acid binding or cell signaling. In the present proposal, n-methylistoic anhydride-derived reagents have been synthesized with affinity to biological molecules, which maintain the property of being fluorescent. The synthesized reagents have two parts; one part is the fluorophore of the n-methylisatoic anhydride and the other is a compound related to one or several biological molecules. These reagents are useful for studies in biochemistry, molecular biology, physiology, microbiology or medicine. And applicable in such different techniques as: cytometry, cellular localization, general protein labeling and specific markings by recognition zones, 1D or 2D electrophoresis, microscopy, anisotropy measurements, DNA transfection or protein quantification. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2683050A1
    申请号:ES201700290
    申请日:2017-03-22
    公开日:2018-09-24
    发明作者:Francisco GARCÍA CARMONA；Samanta HERNÁNDEZ GARCÍA；María Inmaculada GARCÍA GARCÍA
    申请人:Universidad de Murcia；
    IPC主号:
    专利说明:

    The use of fluorescent techniques improves the sensitivity of detection systems, such as 1D and 2D electrophoresis, microscopy or flow cytometry. However, the use of fluorescent techniques is not routinely used in laboratories and is limited to very specific tests due to the high price of the fluorescent reagents used (Sypro Ruby, Atto 488, TAMRA). The price of fluorophores synthesized with n-methylisatoic anhydride is very economical, since the reagents used are very affordable, making it possible to use the compounds synthesized in routine tests. Furthermore, having an excitation wavelength of 275-315 nm, it is possible to visualize the proteins marked on acrylamide gels by simply using an ultraviolet lamp in a dark room.
     10
    Fluorescent labeling is done using molecules called fluorophores, they are molecules that have the ability to emit light when irradiated. In the market there are many fluorescent molecules, such as fluorescein (Bayer, A. (1871).

    "Ueber eine neue Klasse von Farbstoffen". Ber. Dtsch Chem. Ges. 4, 555-558) and its 15 derivatives, fluorescamine (Bóhlen P., S. Stein.W. Dairman, and S. Undenfriend, Arch. Biochem. Biophys. 155, 1973, 213-220), epicoconone (Bell , PJL; Karuso, P. (2003). "Epicocconone, A Novel Fluorescent Compound from the Fungus Epicoccum nigrum." Journal of the American Chemical Society. 125 (31): 9304-9305, [US20060094042A1], [WO2011051225A1]), SYPRO RUBY (Demchenko, A (2011) “Advanced Fluorescence 20 Reporters in Chemistry and Biology III: Applications in Sensing and Imaging Band 3” Advanced Fluorescence Reporters in Chemistry and Biology. Springer), Indiocyanine Green (Benson, RC, & Kues, HA (1978). Fluorescence properties of indocyanine green as related to angiography. Physics in medicine and biology, 23 (1), 159) and their derivatives (Cy2, Cy3, Cy 5), or dansyl chloride (Gray, WR (1967). [12] Dansyl chloride procedure Methods in 25 enzymology, 11, 139-151) among other reagents for biomolecule labeling. In the following invention, methylisatoic anhydride is used as a fluorescent molecule, this molecule has been used in general chemistry for its reactivity [ES2310333, ES8604936, ES0410252], in detection of polysaccharides [CN104535754 (A)] and in enzymatic reactions (Hiratsuka, Toshiaki "New fluorescent analogs of cAMP and cGMP available as substrates for 30 cyclic nucleotide phosphodiesterase." Journal of Biological Chemistry 257.22 (1982): 13354-13358).

    On the other hand, the use of binding techniques for affinity to biomolecules and cells is very widespread, which have been used both for labeling and for the detection, separation and purification of biomolecules and cells. There are many and very diverse techniques of this type, among which the binding of proteins that express histidine tails to nickel complexes (widely used in protein purification) (Hochuli, E., Dóbeli, H., & Schacher, stands out) A. (1987) New metal chelate adsorbent selective for proteins and peptides containing neighborhood histidine residues. Journal of Chromatography A, 411, 177-184), biotin labeling 40 (Diamandis, EP, & Christopoulos, TK (1991). The biotin- (strept) avidin system: principies and applications in biotechnology. Clinical chemistry, 37 (5), 625-636) or antibody labeling (Stirling, JW (1990). Immuno- and affinity probes for electron microscopy: a review of labeling and preparation techniques, Journal of Histochemistry & Cytochemistry, 38 (2), 145-157). Four. Five

    The combination of these two techniques results in an affinity label that can be detected by fluorescence-based instrumentation techniques. Which greatly improves the sensitivity of the system. These types of labels are commercially available and although they are very useful and much more sensitive than conventional detection techniques, their use is limited by their high price.



    Description of the invention

    The present invention relates to a compound of general formula (I), characterized by having a fluorescent part and a part, called R, with affinity for biological molecules (proteins, peptides, antibodies, nucleic acids among others), tissues and cells. 5 These types of compounds constitute an alternative to chemical derivatizations used in proteomics and genomics to introduce biomolecule markers.


     10
    An aspect of the invention is that the fluorescent part of the compound, hereinafter referred to as fluoroforo, forms the n-methylisatoic anhydride. The n-methylisatoic anhydride is a compound widely used in heterocycle chemistry, due to its high reactivity and its reaction mechanism. It is capable of reacting with primary amines, thiols or alcohols under different temperature and pH conditions. In addition to its modest price, it has good fluorescent properties, quantum yield of 0.6, long Stokes displacement (90-150 nm). The compound absorbs ultraviolet light (200-300 nm) and emits blue light (400 nm), which allows it to be detected with commercial equipment.

    In the present invention "fluorophore" is defined as that molecule capable of emitting light when it is irradiated, which can be detected with instrumental techniques such as fluorimetry, spectroscopy, microscopy (both fluorescent and confocal) and image analysis systems.

    The second aspect of the invention is the compound to be reacted with the fluorophore to prepare the labeling reagent. This compound must be able to interact with one or more biological molecules and must meet the following requirements:

    a) that has at least one reactive group (amine, thiol, alcohol) to react with the fluorophore.
    b) that this reactive group does not intervene in the subsequent interaction. 30
    c) that the compound does not reduce or interfere with fluorescent fluorophore capabilities.
    d) that the compound has an interest in the fields of molecular and cellular biology, biochemistry, microbiology or medicine.

    In the present invention "reagent capable of interacting with biological molecules, whole cells or tissues" is defined as any reagent that is capable of binding to a biological molecule or specifically to a characteristic part of the biological molecule or cell (nucleus , membrane, organelles).

    In the present invention "biological molecule" is defined as any molecule present in living organisms (animals, vegetables, fungi, bacteria), more specifically proteins, carbohydrates, lipids, nucleic acids.

    In the present invention, both eukaryotic and prokaryotic cells are referred to as "cells."

    Preferably the compounds capable of interacting with biological molecules that meet the aforementioned requirements are:

    a) Compounds capable of chelating metals, such as iminodiacetic acid of general formula (II), carboxymethyl aspartate of general formula (III) or bis carboxymethyl lysine of general formula (IV) among others, these compounds chelated with metals such as Ni + 2, Cu + 2, Fe + 2 or Co + 2 among others, are capable of binding very selectively to proteins that express histidine tails.



     fifteen



    b) Hydrophobic chains, are chains formed by two or more attached methyl (alkyl chains) that have general formula (V), where R is an alcohol, an acid or an amine, more specifically an amine, have hydrophobicity. Hydrophobic chains have affinity for lipid membranes or surfactants such as SDS.

    CH3- (CH2) n-R (V)
     25
    c) Biotin, of general formula (VI) and its derivatives is a specific marker of proteins such as streptavidin or avidin.


    d) Heparin, of general formula (VII) and its derivatives is a disaccharide with a high affinity for thrombin, a protein present in the blood and responsible for coagulation of it. Heparin also has an affinity for fibronectin. 5 proteins with thrombin tails are also overexpressed as a specific marker.



    e) Antibodies against FLAG-tag, HA-tag, Myc-tag among others. 10

    f) Enzyme inhibitors, with medical or pharmacological interest such as caspase inhibitors, among others.

    g) Polyamides that have a high affinity for nucleic acids. fifteen

    h) Poly ethylene glycols that have affinity for proteins and nucleic acids.

    Another aspect of the invention is the application of these fluorescent compounds for the detection of biological molecules, cells or tissues. As are the detection of specific proteins once electroeluted in agarose or acrylamide gels, preferably acrylamide gels, the detection in liquid medium using fluorescence or fluorescent anisotropy measurements or the marking and detection of cells by fluorescent or confocal microscopy, preferably confocal microscopy. Applicable in fields as diverse as biochemistry, microbiology, molecular and cellular biology, analytics or medicine. 25

    Brief description of the content of the figures

     FIG. 1. Synthesis scheme of an affinity fluorescent reagent (MI-NTANi + 2) to proteins that express histidine tails. 30

     FIG 2. HIS-tag protein electrophoresis gel labeled with the fluorescent reagent MI-NTANi + 2.

     FIG. 3. Synthesis scheme of a hydrophobic fluorescent reagent (MI-C18). 35

     FIG. 4: Protein electrophoresis gel (BSA and lysozyme), stained with the synthesized fluorescent reagent MÍ-C18.

     FIG. 5. Method of titration of proteins in liquid medium, the 96-well plate contains and different concentrations of BSA (0-1 mg / mL). 5

     FIG. 6. Scheme of labeling of lipid membranes with the hydrophobic reagent Ml-C18.

     FIG. 7. Confocal microscopy of a Bacillus subtilis labeled with the prepared hydrophobic reagent MI-C18. 10

    Description of a preferred embodiment of the invention

    Example 1: Synthesis of a fluorescent reagent for the detection of HIS-tag proteins (MI-NTA). fifteen

    To synthesize MI-NTA, 13.1 mg of Nα, Nα-Bis (carboxymethyl) -L-lysine was dissolved in 50 mL of phosphate buffer (20 mM, pH 8.7), the final concentration being in the solution 1 mM. Then 500 µL of n-methylisatoic anhydride (100 mM) dissolved in DMSO was added, the final concentration being 1 mM. The solution was stirred for 4 hours at room temperature. The nickel was charged by adding 12 mg of NiCI2 6 H2O to 50 mL of MI-NTA to prepare MI-NTANi + 2. Figure 1 shows the reaction scheme and subsequent affinity labeling.

    Example 2: Application of the reagent synthesized in Example 1 to the detection of HIS-25 tag proteins in acrylamide gels.

    SDS gels were prepared with 12% acrylamide / bisacrylamide following the normal procedure (Laemmli 1970), the samples were denatured in Laemmli buffer at 100 ° C for 5 minutes. Different volumes of the sample (2.5-25 µL) were loaded into the gel.

    The gels were washed twice with deionized water, fixed 30 minutes with a fixation solution (40% ethanol, 10% acetic acid) and stained with 50 mL of 100 µM of the reagent prepared in example 1 diluted in Tris / 10 mM HCI, pH 8.4. The gels were allowed to stain 35 for 12 hours at room temperature. The staining was then discarded and the gels were washed twice with 50 ml of wash solution (40% ethanol, 10% acetic acid). The gels were visualized directly in a dark room with a 315 nm UV lamp, images of the gels were obtained with the Al 600 equipment using EPI fluorescence with two excitation channels, blue and green to obtain color images. Figure 2 shows a gel 40 in which only proteins expressing HIS-tag have been labeled with the reagent prepared in example 1. Up to 50 ng of HIS-tag protein can be detected with this reagent.

    Example 3: Preparation of a hydrophobic fluorescent reagent MI-C18.
     Four. Five
    To synthesize the fluorescent hydrophobic reagent MI-C18, 134.7 mg of octadecylamine was dissolved in 50 mL of acetone (1% TEA / acetic acid pH 10.0), the final concentration of octadecylamine being in the 10 mM solution. Then 5 ml of n-methylisatoic anhydride (100 mM) dissolved in DMSO was added, the final concentration being 10 mM. The solution was stirred for 4 hours at room temperature. Figure 3 shows the reaction scheme 50.

    Example 4: Staining of acrylamide gels with the hydrophobic reagent MI-C18 synthesized in example 3.

    Acrylamide gels were loaded with different volumes (2-20 µl) of a solution of BSA and lysozyme (5 mg / mL protein) diluted in Laemmli loading buffer. Gel 5 staining was performed in a 40% ethanol and 10% acetic acid solution using 0.1 mM of the reagent synthesized in Example 3. The gels were allowed to stain 45 minutes under orbital agitation, then washed twice with water for 5 minutes and stored in a solution of 5% acetic acid, 10% ethanol, 2% SDS. Figure 4 shows the image of the gel obtained, using UV fluorescence and EPI fluorescence. 10

    Example 5: Method of titration of proteins in liquid medium using the hydrophobic reagent MI-C18.

    A method of titration of proteins in liquid medium is proposed using reagent 15 synthesized in example 3. The calibration line is prepared with 1.5% acetic acid, 0.1% SDS, the reagent prepared in example 3 MI-C18 1.6 µM and BSA (0.05 - 1 mg / mL). The reagents were mixed in a 3 ml spectrophotometric cuvette and the fluorescence was measured with an excitation wavelength of 350 nm and the emission wavelength at 430 nm. Figure 5 shows the 96-well plate that was used to prepare the calibration line. The 20 results show that the method is linear between 0.015 to 1 mg / mL.

    Example 6: Method of cell labeling using the hydrophobic reagent of example 3 MI-C18.
     25
    Bacillus subtilis bacterial strain was grown in 5 mL of Luria Bertani (LB) medium at 30 ° C for 3-6 hours. The cells were centrifuged at 7000 rpm at 4 ° C for ten minutes, then the supernatant was discarded and the pellet was resuspended in 5 ml of sterile saline (0.85% NaCI). For staining, 1 mL of bacteria in saline were mixed with 5 µM of the reagent prepared in Example 3. The samples were incubated cold (4 ° C) for 12-30 hours, then centrifuged and the cell pellet washed with saline solution three times. The samples were stored in saline at 4 ° C.

    A drop of the sample was placed on a microscope slide and covered with a coverslip. Fluorescence images were taken from the samples using a Leica TCS-SP2 inverted spectral confocal microscopy equipped with AOBS, a D-eclipse C1 confocal module and a DXM 1200C refrigerated chamber. Stained samples were excited using the blue diode (408 nm). Figure 6 shows the procedure for staining lipid membranes with the reagent of example 3. Figure 7 shows the confocal microscopy image of a Bacillus stained with the reagent prepared in example 3, MI-C18. 40
    权利要求:
    Claims (15)
    [1]

    1. Fluorescent compounds derived from n-methylisatoic anhydride of general formula (I), for the marking, detection, quantification, location or signaling of biological molecules, tissues and cells. 5

    Where R is a compound with affinity for biological molecules, cells or tissues.
     10
    [2]
    2. Compounds according to claim 1, wherein the compound with biological affinity is a metal complex with histidine tails affinity, the resulting compound being fluorescent and maintaining the affinity for histidines.

    [3]
    3. Compounds according to claim 1, wherein the compound with biological affinity is an alkylated chain with affinity for hydrophobic biomolecules or hydrophobic areas of proteins, the resulting compound being fluorescent and maintaining its hydrophobicity.

    [4]
    4. Compounds according to claim 1, wherein the compound with biological affinity is biotin or one of its derivatives, the resulting compound being fluorescent and maintaining the affinity for streptavidin and its derivatives.

    [5]
    5. Compounds according to claim 1, wherein the compound with biological affinity is heparin or one of its derivatives, the resulting compound being fluorescent and maintaining thrombin affinity. 25

    [6]
    6. Compounds according to claim 1, wherein the compound with biological affinity is an antibody.

    [7]
    7. Compounds according to claim 1, wherein the compound with biological affinity is an enzyme inhibitor.

    [8]
    8. Compounds according to claim 1, wherein the compound with biological affinity is a polyamine.
     35
    [9]
    9. Compounds according to claim 1, wherein the compound with biological affinity is a polyethylene glycol.

    [10]
    10. Use of the compounds of claims 2-9, for the mapping of biological molecules. 40

    [11]
    11. Use of the compounds of claims 2-9, for the detection of proteins in acrylamide or agarose gels.

    [12]
    12. Use of the compounds of claims 2-9, for the detection of proteins and other biological molecules in liquid medium.

    [13]
    13. Use of the compounds of claims 2-9, for labeling, localization and signaling of cells and microorganisms. 5

    [14]
    14. Use of the compounds of claims 8-9, for the detection of nucleic acids in acrylamide or agarose gels.

    [15]
    15. Use of the compounds of claims 8-9, for labeling, signaling location 10 and transfection of nucleic acids.
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    同族专利:
    公开号 | 公开日
    ES2683050B1|2019-06-26|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2016038205A1|2014-09-11|2016-03-17|Université De Lorraine|Fluorescent carbohydrate conjugates, the preparation method and uses thereof|
    CN104535754A|2014-12-10|2015-04-22|南昌大学|Preparation method for N-methylisatoic anhydride rapidly-labeled polysaccharide|
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